Recent progress in clinical islet transplantation with the introduction of the 'Edmonton Protocol' has created enthusiasm for this approach as an effective therapy for highly selected patients with unstable forms of Type I diabetes. The therapy, however, is not suitable for patients in the earliest stages of Type I diabetes because of potential risks associated with anti-rejection therapies. While the risk of malignancy and life-threatening infection has been very low in recent clinical series, complications of severe mouth ulceration, elevated cholesterol, hypertension and the need for two or more organ donors to secure insulin independence emphasize the need for further improvements in the safety and efficacy profile of islet transplantation if it is to be more broadly applied in diabetes. Our Laboratory has focused intense efforts in exploring ways to induce minimal immunosuppression or tolerance in mouse models of islet transplantation. Recently, we have found that an antibody directed against the surface inducible co-stimulatory molecule ICOS (12A8) is highly effective in prolonging islet allograft survival beyond 100 days in approximately half of treated mice. The current proposal is designed to further explore blockade of the ICOS-B7h pathway in control of allograft rejection and autoimmune recurrence after islet transplantation in mice. We believe that this promising finding could be further enhanced by rational combination of anti-ICOS therapy with complimentary approaches that have potential for rapid clinical translatability. More specifically, anti-ICOS will be combined with either donor specific transfusion, CTLA4-1g, CD40 blockade, or with the drug FTY720 to evaluate its effect in preventing islet allograft rejection. The most promising of these approaches will be further tested in the primary and secondary prevention of spontaneous diabetes in NOD mice. We will explore the immunological mechanisms associated with tolerance phenotypes [mice treated with anti-ICOS based therapies] by donor and third party islet and skin graft rechallenge, by thymectomy, or by following the fate of donor-specific T cells in TCR-transgenic models. Furthermore, in vitro co-culture assays and adoptive transfer studies, in addition to islet-kidney graft re-transplantation experiments will be conducted to search for regulatory T cell activity. We will also study our clinical islet transplant patients by flow cytometry and TaqMan quantitative PCR for ICOS expression in peripheral blood and in graft biopsies. We will correlate clinical outcomes with ICOS expression to determine if ICOS monitoring might be a useful tool for prediction of clinical course. If these initial studies show promise, we would plan to further explore anti-ICOS therapies in primate models of islet transplantation within the context of an extended future proposal, with a view to ultimate testing in clinical islet transplant recipients.